Synthesis, Molecular Docking and In Vitro Screening of Some Newly Synthesized Triazolopyridine, Pyridotriazine and Pyridine–Pyrazole Hybrid Derivatives

A series of novel pyridine and fused pyridine derivatives have been prepared starting from 6-(3,4-dimethylphenyl)-2-hydrazinyl-4-(thiophen-2-yl)-pyridine-3-carbonitrile 1 which on treatment with appropriate formic acid, acetic acid/acetic anhydride, benzoyl chloride and/or carbon disulfide afforded the corresponding triazolopyridine derivatives 2–5. Also, treatment of hydrazide 1 with diethyloxalate, chloroacetyl chloride, chloroacetic acid and/or 1,2-dichloroethane yielded the corresponding pyridotriazine derivatives 7–10. Further transformation of compound 1 with a different active methylene group, namely acetyl acetone, diethylmalonate, ethyl cyanoacetate, ethyl benzoylacetate and/or ethyl acetoacetate, produced the pyridine–pyrazole hybrid derivatives 11–15. These newly synthesized compounds (1–15) were subjected to in silico molecular docking screenings towards GlcN-6-P synthase as the target protein. The results revealed moderate to good binding energies of the ligands on the target protein. All the newly prepared products exhibited antimicrobial and antioxidant activity.

In designing a drug candidate, interaction between drug and receptor is mostly understood by employing molecular docking studies. This provides information about the interaction along with predicting the activity and affinity of the targeted drug candidate on the selected receptor [51]. Enzymes involved in the process of biosynthesis of the cell walls of the microbes are considered to be good targets for docking while designing novel compounds such as anti-microbial candidates. In this regard, the enzyme glucosamine-6-phosphate synthase (GlmS, GlcN-6-P synthase, L-glutamine: D-fructose-6P amido-transferase, EC 2.6.1.16) came out to be an attractive target for docking studies in anti-bacterial and anti-fungal drug design [52] as it is involved in the formation of N-acetyl Glucosamine (the core amino sugar) which is an important building block of the fungal and the bacterial cell wall [53,54]. Nicotinonitrile-based analogues have been reported as potent anti-bacterial and anti-fungal agents [55][56][57]. Also, some nicotinonitrile derivatives have been reported as inhibitors of GlcN-6-P synthase [57]. Thus, it was thought to be worthwhile to predict drug-receptor interaction employing in silico molecular docking screenings of the synthesized compounds on the selected target i.e., GlcN-6-P synthase.
In line with our previous work [58][59][60][61] and our observations, we propose the modification of new heterocyclic compounds containing a pyridine nucleus in the hope that they could possess good anti-oxidant ability and anti-microbial activity.
2-Hydrazino-nicotinonitrile 1 upon diazotization with a solution of sodium nitrite in hydrochloric acid yielded the corresponding tetrazolopyridine derivative 6 (Scheme 1). The IR and 1 H-NMR spectra of the latter compound revealed the absence of characteristic signals for the NH and NH 2 groups and its mass spectrum showed molecular ion peak at m/z (%):331 (M + , 37%), 303 (M + -N 2 , 100%) (cf. the Materials and Methods Section).
On the other hand, treatment of 2-Hydrazino-nicotinonitrile 1 with diethyloxalate, chloroacetyl chloride, chloroacetic acid and/or 1,2-dichloroethane yielded the corresponding pyridotriazine derivatives 7-10, respectively (Schemes 1 and 2). The IR spectrum of dioxopyridotriazine derivative 7, for example, showed absorption bands at 3150; 2209; 1713 and 1675 cm −1 for (NH); (CN) and two (C=O) groups respectively. Its 1 H-NMR spectrum showed signals at δ = 2.26, 2.28 for two CH 3 groups, 7.25-7.34 & 7.49-7.61 for Aromatic-H, 7.90 for pyridine-H, and 9.02 for the NH group; and its mass spectrum showed a molecular ion peak at m/z (%):374 (M + , 22%). Upon spectral measurements the isomeric structures of 8 and 9 were deduced (cf. the Materials and Methods Section). The IR spectrum of pyridotriazine derivative 10 displays absorption bands attributed to (NH) and (CN) and its 1 H-NMR spectrum showed a signal at δ = 4.51-4.88 for 2CH 2 groups in addition to two CH 3  2-Hydrazino-nicotinonitrile 1 upon diazotization with a solution of sodium nitrite in hydrochloric acid yielded the corresponding tetrazolopyridine derivative 6 (Scheme 1). The IR and 1 H-NMR spectra of the latter compound revealed the absence of characteristic signals for the NH and NH2 groups and its mass spectrum showed molecular ion peak at m/z (%):331 (M + , 37%), 303 (M + -N2, 100%) (cf. the Materials and Methods Section).
Further transformation of compound 1 with a different active methylene group, namely acetyl acetone, diethylmalonate, ethyl cyanoacetate, ethyl benzoylacetate and/or ethyl acetoacetate, produced the pyridine-pyrazole hybrid derivatives 11-15 (Scheme 2). The 1 H-NMR spectrum of compound 11 displays signals at δ = 2.30, 2.67 for two new CH 3 groups, 5.96 for pyrazole-H in addition to two CH 3 group, Aromatic-H and pyridine-H (cf. the Materials and Methods Section). The IR spectrum of compound 12 revealed absorption bands attributed to (NH); (CN) and two (C=O) groups at 3220; 2215; 1729 and 1680 cm −1 , respectively, and its 1 H-NMR spectrum showed the signals of CH 2 and NH protons for the pyrazole ring at δ = 5.13 and 9.08, respectively, in addition to two CH 3 group, Aromatic-H and pyridine-H (cf. the Materials and Methods Section). The IR spectrum of compound 13 showed bands at 3233; 3117; 2210 and 1685 cm −1 for (NH 2 ); (NH); (CN) and (C=O) groups, respectively, and its 1 H-NMR spectrum showed the signals of CH; NH and NH 2 protons for the pyrazole ring at δ = 6.12; 7.89 and 9.13, respectively, in addition to two CH 3 group, Aromatic-H and pyridine-H (cf. the Materials and Methods Section). The IR spectra of both 14 and 15 showed bands for (CN) and (C=O) groups at 2217; 1665 and 2215; 1778, respectively. The 1 H-NMR spectrum showed the signals of CH 2 for the pyrazole ring at δ = 2.93; 3.10 and 3.71; 3.96, respectively (cf. the Materials and Methods Section).

In Silico Molecular Docking Screenings
In silico molecular docking screenings were performed after achieving synthesis and characterization of the compounds. GlcN-6-P synthase was considered as the target receptor. In order to determine the best in silico conformation, comparative and automated docking studies were performed with the newly synthesized drug candidates. All the synthesized compounds (1-15) were subjected to docking studies on the target protein GlcN-6-P synthase. Figure 1 shows the docked images of these newly synthesized ligand drug candidates on the target protein. The minimum binding energies and estimated inhibition constants of the synthesized compounds are documented in Table 1. The results of in silico studies revealed a moderate to good pattern of affinity and activity of the synthesized compounds on the target protein which is supported by in vitro antimicrobial screenings as the compounds were active against one and/or the other tested microorganisms. Compounds with higher binding energies came out to be more potent than the standard drug taken. Their binding energies ranged from −6.08 to −7.84 kJ/mol with an estimated inhibition constant ranging from 1.79 to 35 micromol.

In Silico Molecular Docking Screenings
In silico molecular docking screenings were performed after achieving synthesis and characterization of the compounds. GlcN-6-P synthase was considered as the target receptor. In order to determine the best in silico conformation, comparative and automated docking studies were performed with the newly synthesized drug candidates. All the synthesized compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) were subjected to docking studies on the target protein GlcN-6-P synthase. Figure 1 shows the docked images of these newly synthesized ligand drug candidates on the target protein. The minimum binding energies and estimated inhibition constants of the synthesized compounds are documented in Table 1. The results of in silico studies revealed a moderate to good pattern of affinity and activity of the synthesized compounds on the target protein which is supported by in vitro antimicrobial screenings as the compounds were active against one and/or the other tested microorganisms. Compounds with higher binding energies came out to be more potent than the standard drug taken. Their binding energies ranged from −6.08 to −7.84 kJ/mol with an estimated inhibition constant ranging from 1.79 to 35 micromol.   14 and GlcN-6-P.

Examination of the Structural Activity Relationship (SAR)
The excellent antioxidant activity of the synthesized compounds 14 and 15 depends on the electronic environment and structural skeleton of the molecule. The promising activity of these synthesized motifs is mainly due to the introduction of the 5-oxo-3-Phenyl/Methyl-dihydropyrazole ring into the skeleton.It was observed that replacement on the 3rd position in the dihydropyrazole ring by the phenyl ring or the methyl group dramatically enhances the anti-oxidant activity. Furthermore, it is apparent that compound 1 and compound 6 bearing the hydrazide and tetrazole group, respectively, at the periphery of the molecular chain, have moderate antioxidant activity. The test was done using the diffusion agar technique, well diameter: 6.0 mm (100 µL was tested). NA: No activity. All sample at 5 mg/mL.

General Information
Melting points were measured using an Electro-Thermal IA 9100 digital melting point apparatus (Büchi, Flawil, Switzerland) and are uncorrected. Infrared spectra were recorded on a Perkin-Elmer 1600 FTIR (Perkin-Elmer, Waltham, MA, USA) discs. NMR spectra were determined on a Jeol-Ex-500 NMR spectrometer (JEOL, Tokyo, Japan) and chemical shifts were expressed as part per million; (δ values, ppm) against TMS as internal reference, National Research Center, Cairo, Egypt. The mass spectra were run at 70 eV with a Finnigan SSQ 7000 spectrometer (Thermo Electron Corporation, Madison, WI, USA) using EI and the values of m/z are indicated in Dalton. Elemental analyses were performed on a Perkin-Elmer 2400 analyzer (Perkin-Elmer) and were found within the accepted range (± 0.30) of the calculated values. Reaction monitoring and verification of the purity of the compounds was done by TLC on silica gel pre-coated aluminum sheets (type 60 F254, Merck, Darmstadt, Germany). All solvents and chemical reagents were purchased from Aldrich (Munich, Germany). Compound 1 was prepared according to a reported method [62].

In Silico Molecular Docking Screenings
A Lamarckian genetic algorithm contained in AutoDock (version 4.0) was employed for in silico molecular docking studies [65] as it is more reliable, efficient, successful and possesses more degree of freedom for ligands as compared to the Monte Carlo method present in old versions of AutoDock. ChemSketch was used for drawing the ligands and that file format was converted into PDB format using Open Babel [66]. Glucosamine-6-phosphate of GlcN-6-P synthase (PDB ID 2VF5) was obtained from Protein Data Bank (http://www.pdb.org/pdb/home/home.do) with the best accurate active region as solved by experimental crystallographic data [67]. This protein was then optimized by removing all the heteroatoms, rotating all the torsions and adding the C-terminal oxygen. Its energy was minimized using by PRODRG server [68]. Ligand was added with polar hydrogen's along with assignment of Kollman partial charges. We made and adjusted the grid in X, Y, Z-axis so as to cover the entire active site of protein i.e., the 12 amino acids residues (Cys300, Gly301, Thr 302, Ser 303, Ser 347, Gln 348, Ser 349, Thr 352, Val 399, Ala 400, Ala 602 and Lys 603) by setting the grid box size at 70, 64, and 56 Å for x, y and z, respectively, and the grid center to 30.59, 15.822 and 3.497 for x, y and z, respectively, with grid spacing of 0.375 Å. Standard protocol was undertaken for docking studies. The docking results were interpreted according to the .pdb file. The co-ordinates of the minimum energy run were determined using the rmsd table created in the .dlg file. UCSF Chimera 1.11.2 was used to visualize this ligand protein interaction within region of 6.5 Å.

In Vitro Anti-Microbial Screenings
The Susceptibility Tests were performed according to National Committee for Clinical Laboratory Standards (NCCLS) recommendations (1993). Screening tests regarding the inhibition zone were carried out by the well diffusion method according to Hindler's method [63]. The inoculum suspension was prepared from colonies grown overnight on an agar plate and inoculated into Mueller-Hinton broth (fungi using malt broth). A sterile swab was immersed in the suspension and used to inoculate Mueller-Hinton agar plates (fungi using malt agar plates). The compounds were dissolved in DMSO with different concentrations (10, 5, 2.5 mg/mL). The inhibition zone was measured around each well after 24 h at 37 • C. Controls using DMSO were adequately done.

DPPH Radical Scavenging Activity
Freshly prepared (0.004% w/v) methanol solution of DPPH radical was prepared and stored at 10 • C in the dark. A methanol solution of the test compound was prepared. A 40 uL aliquot of the methanol solution was added to 3 ml of DPPH solution. Absorbance measurements were recorded immediately with a ultraviolet (UV)-visible spectrophotometer (Milton Roy, Spectronic 1201). The decrease in absorbance at 515 nm was determined continuously, with data being recorded at 1 min intervals until the absorbance stabilized (16 min). The absorbance of the DPPH radical without anti-oxidant (control) and the reference compound ascorbic acid were also measured. All the determinations were performed in three replicates and averaged. The percentage inhibition (PI) of the DPPH radical was calculated according to the formula: where A C = Absorbance of the control at t = 0 min and A T = absorbance of the sample + DPPH at t = 16 min.

Conclusions
This study focused on the synthesis of new nicotinonitrile derivatives which were synthesized and characterized using spectral and elemental analyses. All synthesized compounds 1-15 were screened for anti-oxidant activity. Compounds 14 and 15 showed excellent anti-oxidant activity at all concentrations due to the introduced 5-oxo-3-Phenyl/Methyl-dihydropyrazole ring. Furthermore, it is apparent that compound 1 which has a hydrazide group at the periphery of the molecular chain has moderate anti-oxidant activity. In silico studies were undertaken to predict the activity and affinity of the synthesized compounds towards the target protein i.e., Glucosamine-6-phosphate synthase and the results were reported to be ranging from −6.08 to −7.84 kJ/mol along with estimation of inhibition constant, Ki, from 1.79-35 micromol. Also, the prepared compounds were tested for their preliminary in vitro anti-microbial activity against different microorganisms. Some of the compounds came up as active against screened bacterial and fungal strains while other were non-reactive. The in silico and in vitro results are consistent with each other as some of the synthesized compounds have demonstrated moderate to good anti-bacterial and anti-fungal activities as compared to the standard reference drugs. Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.